Protein aggregation and amyloidogenesis have been associated with several neurodegenerative disorders like Alzheimer’s, Parkinson’s etc. Unfortunately, there are still no proper drugs and no effective treatment available. Due to the unique properties of noble metallic nanoparticles, they have been used in diverse fields of biomedicine like drug designing, drug delivery, tumour targeting, bio-sensing, tissue engineering etc. Small-sized silver nanoparticles have been reported to have anti-biotic, anti-cancer and anti-viral activities apart from their cytotoxic effects. The current study was carried out in a carefully designed in-vitro to observe the anti-amyloidogenic and inhibitory effects of biologically synthesized green silver nanoparticles (B-AgNPs) on human serum albumin (HSA) aggregation taken as a model protein. We have used different biophysical assays like thioflavin T (ThT), 8-Anilino-1-naphthalene-sulphonic acid (ANS), Far-UV CD etc. to analyze protein aggregation and aggregation inhibition in vitro. It has been observed that the synthesized fluorescent B-AgNPs showed inhibitory effects on protein aggregation in a concentration-dependent manner reaching a plateau, after which the effect of aggregation inhibition was significantly declined. We also observed meaningful chaperone-like aggregation-inhibition activities of as-synthesized florescent B-AgNPs in astrocytes. Several neurodegenerative chronic disorders like Alzheimer’s, Parkinson’s, systemic amyloidosis, etc., involve the aggregation and deposition of misfolded proteins ranging from small oligomers to large amyloid masses ,2. AmyloNowadays, nanotechnology is gaining the focus of major scientific communities due to its broad applicability. It is a multidisciplinary scientific field that is witnessing rapid growth. Despite its enormous scope, one of nanotechnology’s most significant benefits will be the development of innovative and successful medical treatments. Understanding the interactions of nanomaterials and biomolecules both in extracellular and inside the cells will open up new strategies for developing effective nanomedicines ,10,11. NRosa petals extracts by spectroscopy, dynamic light scattering (DLS), and microscopy. Then, we elucidated the mechanism through which B-AgNPs prevent protein aggregation in a concentration-dependent manner in vitro. The role of biogenic AgNPs in inhibiting aggregation was investigated by carefully controlling the artificial environment on the aggregation pathway of human serum albumin (HSA) using several assays such as circular dichroism (CD), ANS fluorescence, Thioflavin T (ThT) fluorescenceCongo red (CR) assay, and turbidity assay. Additionally, we examined the uptake of these B-AgNPs in astrocytes to determine their ability to suppress protein aggregation under in vitro conditions.In the present study, we first characterized the bio-synthesized green AgNPs, (referred as B-AgNPs) using The Rosa petal extract (AgNO3). The synthesis was carried out by incubating a fixed volume of silver nitrate (1 mM AgNO3) with various quantities of the OS stock (150–500 µL) [g for 10 min to remove the unwanted plant products/materials. Finally, the pellet was lyophilized to obtain the nanoparticles. The synthesized B-AgNPs were then characterized employing spectroscopy, Transmission electron microscope (TEM) [TEM, H-7500, Hitachi Ltd., Tokyo, Japan and Dynamic light scattering  techniques for their shape and size analysis.B-AgNPs were synthesized via reducing the silver precursor, silver nitrate, mediated by –500 µL) ,17. DeioThe UV-Vis absorption spectra of silver ions were examined to determine their biogenic reduction to colloidal nanostructures (B-AgNPs) . UV-VisiThe hydrodynamic particle size was studied in Beckman Coulter Delsa Nano particle size analyzer   at ambieStock solution (5 mg/mL) of HSA was prepared in a sodium phosphate buffer of pH 7.4 and then left overnight for dialysis. Lowry’s assay was used for determining the concentration of protein stock solution ,22. DetaThe preliminary conformational change analysis in the native HSA in 5 M urea and increasing concentrations of B-AgNPs was done by performing a turbidity assay . The met16H13NO3S) is a dye that binds to the hydrophobic regions of a polypeptide [ANS  has been used for decades as the gold standard for detecting amyloid fibrils in solution and tissue secretions. Spectro fluorophotometer  was used for the ThT spectral analysis. Quartz cuvette of path length 1 cm was used. Excitation was done at 440 nm and emission spectra were recorded at 450–600 nm. Double distilled water (pH 7.0) was used for the preparation of ThT solution. The final concentration of HSA used was 5 μΜ while that of ThT was 20 μΜ. Also, fluorescence microscopy was used to visualize the fluorescence associated with ThT-bound HSA amyloid. ThT possesses excitation and emission spectrum peaks at around 450 and 490 nm, respectively, responsible for the observed green fluorescence. Briefly, the amyloids from different groups were taken and plated on a coverslip glass on a glass slide and observed under Flourescence microscopy (FM) for the FITC channel  [g for 15 min. The pellets obtained were incubated with 20 µM ThT for 30 min at room temperature and then transferred onto a glass slide to be analyzed under fluorescence microscope and images were observed at 10X magnification . [The binding of Thioflavin T (ThT) dye with β-amyloid gives characteristic spectra which has been used for unfolding mediated aggregation of proteins for many years. ThT  ,28, In a Japan). ,30.32H22N6Na2O6S2) is a dye that binds to β-amyloid of aggregated proteins and causes a redshift in the spectra as compared to the native proteins. UV-Vis1700, spectrophotometer , was used for taking CR absorbance measurements in the range of 400–700 nm using a quartz cuvette of path length 1 cm. Like ThT analysis, the CR based imaging was also performed for the different groups and slides were observed under FM for the red fluorescence channel (TriTC) at 10X magnification [Congo red  were plated on a coverslip on a 6 well plate at 37 °C in a humidified atmosphere at 5% CO2 were treated with variable B-AgNPs concentration for 2 h. After 2 h of incubation, the cells were washed with sterile PBS (pH 7.4) to remove excess AgNPs. After washing, the astrocytes were observed in a fluorescent microscope  at 10X to check their uptake.Astrocytes -2,5-diphenyltetrazolium bromide) assay was used to investigate the toxicity of HSA aggregate on astrocyte cells. In 96-well plates, cells were grown in DMEM-F-12 (10% FBS) medium until they reached 70% confluency. Cells were rinsed with sterile PBS and then treated for 24 h at 37 °C in 5% COp < 0.05 was considered [All of the tests were carried out in triplicate and the results were expressed as the standard error mean. Graph Pad Prism 7.0 was used to analyze the data after the one-way analysis of variance . The statistically significant difference at nsidered .Rosa flower extract to synthesize silver nanoparticles. Rosa Santana is a prominent ornamental Rosaceae cultivar. The flower is rich in tannin and pectin, as well as fatty acids and organic acids [In our studies, we used ic acids . Additioic acids . Due to The synthesis of silver nanoparticles in aqueous petal extract was investigated using absorption spectra spanning 300–700 nm wavelength. The shift in colour of the silver nitrate solution to blackish-brown with the addition of petals extract was interpreted as evidence for the creation of silver nanoparticles. At 432 nm, a single high signal was found in the spectra, confirming the production of the silver nanoparticles A. The UVw/v) when the protein samples were incubated at a temperature mimicking the pathological temperature during fever, i.e., 40 °C in vitro. In the current study, we discovered that B-AgNPs inhibited protein aggregation in a concentration-dependent manner. Still, the maximum inhibition was observed at 35 μg/mL (w/v), above which the effect of protein aggregation inhibition was reduced or became insignificant, as indicated by turbidity assay measurements (w/v) B-AgNPs. Above 35 μg/mL (w/v) B-AgNPs, the incubated samples showed slightly increased turbidity, which might be due to decreased inhibition of protein aggregation induced by urea above that concentration of B-AgNPs. ANS fluorescence measurements further analyzed changes in the native conformations and aggregation inhibition in HSA by B-AgNPs. It is well established that ANS binds with the hydrophobic residues of the polypeptides, which is exposed during the unfolding of a native protein. ANS spectra also supported our findings that B-AgNPs inhibited protein aggregation in a concentration-dependent manner reaching its maximum at 35 μg/mL (w/v) B-AgNPs at 37 °C. w/v) B-AgNPs, at which point the fluorescence intensity increased. This demonstrated that the highest inhibition of protein aggregation occurred at a concentration of 35 µg/mL (w/v) of B-AgNPs, above which the effect of protein aggregation inhibition was diminished, perhaps due to B-AgNPs self-aggregation. However, when we incubated the HSA with varying concentrations of B-AgNPs in the absence of urea, the nanoparticles doesn’t showed prominent changes in the native conformations of HSA and aggregation is not observed as was observed in the presence of 5 M urea [We initially examined the structural changes in native HSA and HSA incubated with increasing concentrations of B-AgNPs in the presence of 5 M urea using turbidity measurements at 360 nm. The study aimed to analyze the effect of B-AgNPs on the aggregation of HSA induced by urea at physiological temperature, i.e., 37 °C, in critically designed in vitro conditions. Our previous study found that the B-AgNPs (size 10–50 nm) inhibited protein aggregation induced by urea maximally at 30 μg/mL (urements A. The tu5 M urea .Circular dichroism (CD) spectroscopy is a frequently used technique for quantifying the secondary structure and unfolding of proteins ,42. The w/v), above which no appreciable aggregation inhibition was seen. Although this is not a confirming test for protein aggregation and inhibition of protein aggregation, but it provides the most plausible explanation for the phenomena mentioned above, which was confirmed by many experiments.Far-UV CD spectral measurements were used to investigate changes in the secondary structure of native HSA. Far-UV CD spectral analysis provides information about a protein’s secondary structure changes . The natw/v) B-AgNPs, above which the effect was considerably reduced. Similar to ANS assay, we performed ThT assay, where we incubated the HSA in the presence of increasing concentrations of B-AgNPs without 5 M urea. The ThT and ANS spectral analysis in the absence of 5 M urea doesn’t show noticeable impact on the native conformation of protein, also aggregation is not noticed as was observed with 5 M Urea [The dye thioflavin T (ThT) is widely regarded as the gold standard for detecting protein aggregates . It has 5 M Urea .w/v) compared to untreated, which shows all green-fluorescent aggregates (w/v) B-AgNPs, above which there was significant decrease in the protein aggregation inhibition by B-AgNPs. Like, ThT based microscopic analyses we also carried out image analysis for the B-AgNPs based inhibition for amyloidal aggregate formation after B-AgNPs incubation for CR based dye. The data observed under fluorescence microscopy reciprocates the results observed under spectroscopy, suggesting its rationality as a chaperone agent  when HSA was co-incubated with B-AgNPs at 35 μg/mL (gregates B. For fugregates A. The enne agent B.w/v) of B-AgNPs (w/v), as demonstrated here with 55 µg/mL (w/v) of B-AgNPs  B-AgNPs treated amyloidal aggregates. This finding confirms that B-AgNPs’ anti-amyloidogenic activity increased cell viability. ANOVA was utilized to analyze the MTT results (To determine the amount of HSA-amyloidal aggregate cytotoxicity, the MTT assay was used. The HSA amyloidal aggregates treatment leads to around 62% viability in the cells. Suggesting, HSA amyloidal aggregates have cellular toxicity. In contrast, the cell viability was enhanced to 70% and 82% respectively, in the presence of 5 μg/mL and 35 μg/mL ( results B. We hypThe depletion of hazardous aggregates by B-AgNPs results in a reduction in astrocytes toxicity. Additionally, incubation of B-AgNPs with astrocytes resulted in their uptake into the cells, as evidenced by the presence of fluorescing B-AgNPs within the cells A. Once wThe present work demonstrated the effect of different concentrations of B-AgNPs, on the putative conformational changes in HSA. AgNPs have seen a dramatic surge in application in the pharmaceutical and food industries due to their long-known multifunctional properties. That is why it is critical to examine the effect of nanoparticles on macromolecules’ shape and stability. We believe that the synthesized smaller size B-AgNPs may be beneficial in suppressing protein aggregation up to a concentration of roughly 35 µg/mL. Above this point, the inhibitory impact diminishes. As a result, the use of smaller nanoparticles requires additional in vivo testing and should be carefully evaluated. The study is a preliminary examination to determine the influence of B-AgNPs on the inhibition of aggregation of HSA in the presence of urea. One could argue that the prevailing conditions in natural systems are significantly more complicated, with a diversity of proteins and other factors interacting with the protein under study. However, the current study can serve as a springboard for future efforts to establish an effective technique for preventing the protein-associated amyloid formation and unravel metal nanoparticles’ role as a potent inhibitor of amyloid/fibril formation. This study will aid in the design of in vivo experiments to determine the effect of B-AgNPs on animal models, which will aid in the development of more effective drugs for aggregation-related disorders and will also serve as a guide for the pharmaceutical and food industries in terms of limiting their use of AgNPs. Our findings may pave the way for nanoparticle-based therapies for disorders associated with protein aggregation.


M. leprae DNA was detected. In 63.1% of fingerstick blood samples, M. leprae specific antibodies were detected with the quantitative αPGL-I test. Results show a strong correlation of αPGL-I IgM levels in fingerstick blood and RLEP-qPCR positivity of nasal swabs, with the M. leprae bacterial load measured by RLEP-qPCR of skin biopsies. Patients with a high bacterial load  can be identified with combination of counting lesions and the αPGL-I test. To our knowledge, this is the first study that compared αPGL-I IgM levels in fingerstick blood with the bacterial load determined by RLEP-qPCR in skin biopsies of leprosy patients. The demonstrated potential of minimally invasive sampling such as fingerstick blood samples to identify high bacterial load persons likely to be accountable for the ongoing transmission, merits further evaluation in follow-up studies.The World Health Organization (WHO) endorsed diagnosis of leprosy  entirely based on clinical cardinal signs, without microbiological confirmation, which may lead to late or misdiagnosis. The use of slit skin smears is variable, but lacks sensitivity. In 2017–2018 during the ComLep study, on the island of Anjouan , of whom 263 were sampled for a skin biopsy and fingerstick blood, and 260 for a minimally-invasive nasal swab. In 74.5% of all skin biopsies and in 15.4% of all nasal swabs,  M. leprae (αPGL-I IgM) in fingerstick blood correlates well with the bacterial load determined in skin biopsies from the same leprosy patient. Therefore, a high level of antibodies against M. leprae in fingerstick blood might identify persons who pose a potential risk for transmission of leprosy and could be prioritized for contact screening, which is essential for control of the disease.Leprosy is the oldest infectious disease known to humankind. We still do not succeed in curbing its transmission, with more than 200,000 new patients detected worldwide each year. Identifying persons with a high burden of bacteria is key to curb transmission. To identify these persons, bacteria are counted in invasive and painful samples like slit skin smears and skin biopsies. We evaluated whether we can use less invasive samples, like fingerstick blood or nasal swabs, to determine the bacterial load. We found that the level of antibodies against  According to the World Health Organization (WHO) the global leprosy  prevalence has decreased to <1 patient per 10,000 population since the year 2000, based on which leprosy is eliminated as a public health problem. However, the annual global incidence has stabilized since 2006, with approximately 200,000 new leprosy patients reported worldwide each year )/36 RLEP copy numbers.Quantification was done by adding to each qPCR run a serial dilution of v/v) Triton X-100  BSA) to lyse blood cells. The diluted fingerstick blood sample (50μl) was flowed on lateral flow strips the same day after transportation to the central laboratory at ambient temperature. The lateral flow strips were transported to LUMC. By using an anti-IgM UCP reporter conjugate, the human αPGL-I IgM antibodies were detected as described in Corstjens et al. (2019) [4:YB3+, Er3+ polyacrylic-acid coated nano-sized particles, 980 nm excitation and 550 nm emission) were obtained from Intelligent Material Solutions, Inc. . A Packard FluoroCount microtiterplate reader compatible with the UCP technology [relative fluorescence units (RFUs) measured at the respective lines. For this cohort, the αPGL-I UCP-LFA the R-value threshold was set at 0.29 according to and determined as described in previous studies [The capillary fingerstick blood collected with a disposable 20 μl Minivette collection tubes , was diluted 1:50 by immediate mixing with 980μl assay buffer. The buffer was supplemented with 1% (. (2019) . UCP mat studies .M. leprae Thai 53) and a negative extraction control (water) were extracted in parallel with the clinical samples in each run to check extraction performance and to rule out contamination during the extraction procedure. An internal qPCR control, a positive and negative (water) DNA qPCR control were run simultaneously with the samples to check performance of the qPCR and to rule out DNA contamination during the qPCR procedure.For molecular analyses, on each sampling day, (negative) environmental control swab samples were collected, to rule out contamination during sampling. A positive  control sera were included for the αPGL-I test.Data from the assays were log transformed . Data obtained for the different laboratory assays were evaluated using parametric hypothesis tests. Comparing a quantitative result between the two groups was carried out using the Welch t-test. The difference between months on treatment and the binary outcome of assays, was evaluated with the non-parametric Mann-Whitney U test. The alternative hypothesis stating significant differences between two outcomes was accepted at a significance level of α = .05.The multiple regression models modelling bacterial load versus αPGL-I R-values used the 10-based logarithm of the bacterial load (determined by RLEP-qPCR) as dependent variable, with the 10-based logarithm of the R-values of αPGL-I test as the independent, continuous variable, and a grouping variable splitting the population into 3 groups: i) patients with a negative nasal swab and less than 25 lesions, ii) patients with either a positive nasal swab or ≥ 25 lesions, iii) patients with both a positive nasal swab and ≥ 25 lesions. To analyse whether the effect of R-value of the αPGL-I test on the bacterial load was uniform across the 3 groups, the interaction between αPGL-I R-values and nasal swab group was tested for significance. The inclusion of the different factors in the multiple regression model was based on a simple linear regression, modelling the effect of each factor on the log10. Significant factors were entered into a multiple linear regression model . SubsequPatients having ≥50,000 bacilli in a 4mm biopsy were further referred to as high bacterial load (HBL) patients. To evaluate if it was possible to predict the odds of being a HBL-patient, sensitivity and specificity were calculated for the three independent variables  separately. For the presence of ≥25 lesions, for the nasal swab positivity by RLEP qPCR and for the continuous αPGL-I R-value, a ROC-curve was constructed, and the optimal cut-off value was determined by the Youden index , which wAll analyses were conducted with R version 3.5.0 for Windows .From January 2017 until January 2018, 310  leprosy patients were diagnosed. For 263 (84.8%) patients, sampling was complete (fingerstick blood samples and skin biopsies), with 260 providing a nasal swab from both nostrils instead of a nasopharyngeal swab. Based on clinical examination, 117 patients were classified as paucibacillary and 146 as multibacillary leprosy. Of these, 137 multibacillary patients provided a slit skin smear, of which 62 had a BI >0. At the time of sampling, 53 of the patients  had received no treatment, versus 210  who started their treatment . Female M. leprae DNA with the RLEP-qPCR (M. leprae DNA in their skin biopsy (M. leprae DNA was detected (98.4%). For 9 multibacillary patients no slit skin smear, and therefore no BI was available  tested positive for LEP-qPCR . Of the LEP-qPCR . As expen biopsy . In all vailable . There wM. leprae DNA with the RLEP-qPCR  tested positive for LEP-qPCR . The bacLEP-qPCR . Of the sal swab , comparesal swab . There wFingerstick blood samples of 166/263 (63.1%) patients tested positive for αPGL-I, including 62/117 (53.0%) paucibacillary patients and 104/146 (71.2%) multibacillary patients . The levTo study the relationship between the αPGL-I IgM levels  and the bacterial load in skin biopsies, we fitted a multiple linear regression model, including 263 patients . The patM. leprae DNA. The 29 HBL-patients with a negative result for the nasal swab tended to have taken treatment longer at the time of sampling than the nasal swab positive HBL-patients (p = 0.03006). Of the same 64 HBL-patients, 57 provided a slit skin smear, including 52 (91.2%) with a mean BI-value >0  patients. Of all the 64 HBL-patients that provided a fingerstick blood sample, 63 tested positive for αPGL-I test , and 60 of those had a αPGL-I R-value ≥0.81. All HBL-patients except one provided a nasal swab, of which 34 (54.0%) contained value >0 . Out of value >0 .The separate analysis of the three independent variables indicates that based solely on the clinical feature (≥25 lesions) a HBL-patient can be identified with 65.5% sensitivity and 92.4% specificity. The highest sensitivity (93.8%) for identifying a HBL-patient is obtained at αPGL-I R-value ≥0.81, however with a reduced specificity of 80.9%. The nasal swab result as predictor for HBL has the lowest sensitivity (54.0%) with the highest specificity (97.0%) . CombiniM. leprae bacterial load as measured by RLEP-qPCR in skin biopsies. To our knowledge this is the first assessment of the correlation of αPGL-I levels with a quantitative molecular measurement in a skin biopsy taken as a proxy for the bacterial load in a leprosy patient. Although a precise determination of the bacterial load by αPGL-I IgM levels is not possible, a range can be estimated. In 2019 Corstjens et al [et al [Our findings confirm a strong correlation between αPGL-I IgM levels and the ns et al  demonstret al [M. leprae infection rather than disease. The advantage of using the αPGL-I test on fingerstick blood to support clinical diagnosis, is that this test is minimally invasive, user-friendly and can be performed in remote laboratories such as in the Union of the Comoros, where no specialized facilities are present. Although the RLEP-qPCR on skin biopsies has overall better sensitivity than the αPGL-I test on fingerstick blood and RLEP-qPCR on nasal swab, the downside of using skin biopsies as a confirmation method, is the invasiveness of the sampling, for which local anaesthesia is necessary and a scar remains. Moreover, to avoid false positives, careful processing of the samples in advanced molecular laboratories needs to be ensured.The RLEP-qPCR on skin biopsies was able to confirm 74.5% of all clinically diagnosed leprosy patients, 80.1% of the multibacillary patients, and 98.4% of all BI positive patients. The RLEP-qPCR on nasal swab detected only 15.4% of all leprosy patients, 25.7% of the multibacillary patients, and 60.7% of all BI-positive patients, which is in line with previous data in an Asian cohort , yet is et al . The αPGUntreated leprosy, particularly lepromatous cases, can lead to serious irreversible nerve damage, often resulting in significant disfigurement and disabilities. Treatment is usually based on the spectral phenotype of the disease. Leprosy presents as a spectral disease, which is more complicated than paucibacillary/multibacillary determination, as demonstrated by the Ridley-Jopling classification for which histopathology is crucial . The WHOM. leprae transmission has been demonstrated several times [et al. found that patients with a positive BI were four times more likely to transmit the disease to their contact in comparison with multibacillary patients with a negative BI and eight times more likely with a BI>3 [M. leprae infection rather than disease, our data confirm that in this cohort in the Union of the Comoros high αPGL-I IgM levels are indicative for a higher bacterial load, which is in line with findings of van Hooij et al. in 2017 [M. leprae infection, while a αPGL-I threshold of 0.81 (in addition to counting lesions) would allow to identify HBL-patients who may pose an increased risk for transmission. Adding the nasal swab, which may also be transiently positive, does not result in extra power for identifying HBL-patients. In the PEOPLE study  ongoing investigation for (close contact) secondary cases will allow to test the hypothesis that high risk index cases of M. leprae can be identified with non-invasive sampling. This will help resolve whether they should be prioritized for extensive contact screening beyond the household, to possibly provide prophylactic treatment to these contacts.For leprosy it is extremely difficult to identify high risk index cases based on secondary cases as the incubation period of leprosy is exceptionally long (on average 5 years and can take up to 20 years or longer). Identification of high risk index cases is key to curb transmission, and therefore this remains an important knowledge gap, as identified during the COR-NTD conference . That multibacillary patients are primarily responsible for al times –27 and Sh a BI>3 . To obta in 2017 , where tOur patients were sampled at variable intervals since the start of treatment limiting our ability to determine diagnostic sensitivity at baseline for the different tests. To control for potential bias, we added this variable to the regression models as a covariate. However, it was nowhere significant, neither in the full model with the interaction between antibodies response and different groups, nor in the linear regression models for the three separate groups (results not shown). None of the conclusions regarding significance and effect sizes were altered by adding months of treatment prior to sampling to the models. Therefore, the month of treatment was not included as a covariate in any of the models, and it is very unlikely that different treatment intervals would bias the results.In this study we lack a non-exposed population control for the αPGL-I test, although we incorporated (non-)endemic controls sera. Inclusion of more non-diseased endemic controls may allow to identify titers indicative of (multibacillary) disease versus latent infection.M. leprae can confirm transmission chains. Correlation with our findings on the bacterial burden may help identify high risk index cases and their characteristics, and allow us to test whether leprosy patients with higher outcome in the αPGL-I test, e.g. αPGL-I test R-value above 0.81, have significantly more secondary cases than patients with lower αPGL-I levels in a certain time frame. The ongoing adaptation of the αPGL-I, full integration of the test in an individually wrapped cassette and improved availability of readers will facilitate actual Point-of-Care testing.The extension of the ComLep cohort in the ongoing PEOPLE study, including door-to-door screening for leprosy in highly endemic villages allows to identify the best predictors of transmission to close contacts. Additionally, the genotypic comparison of M. leprae utilizing less invasive sampling are desired and shown here to be feasible. The bacterial load is not routinely measured for leprosy patients, although it is a known correlate of infectiousness [In conclusion, improved approaches for microbiological confirmation of iousness . CountinS1 FigPB = paucibacillary according to the operational WHO classification; *MB = multibacillary according to the operational WHO classification.(DOCX)Click here for additional data file.S1 TableThe inclusion of the different factors in the multiple regression model was based on a univariable analysis for each of the factors, estimating the influence of a factor to the bacillar load in the skin biopsy.(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 TablePB = Paucibacillary operational WHO classification; MB = Multibacillary operational WHO classification.(DOCX)Click here for additional data file.


Graphene oxide (GO) is extensively studied as a template material for mesenchymal stem cell application due to its two-dimensional nature and unique functionalization chemistries. Herein, a new type of peptide-conjugated multilayer graphene oxide (peptide/m-GO film) was fabricated and used as biomaterial for culturing human Wharton’s jelly-derived mesenchymal stem cells (WJ-MSCs). The characterization of the peptide/m-GO films was performed, and the biocompatibility of the WJ-MSCs on the peptide/m-GO films was investigated. The results demonstrated that the peptide conjugate on the m-GO film did not hamper the normal growth of WJ-MSCs but supported the growth of WJ-MSCs after the 6-day culture period. In addition, the osteogenic differentiation of WJ-MSCs on the peptide/m-GO films was enhanced as compared with the parent m-GO film. Therefore, such peptide-conjugated m-GO films could provide a highly biocompatible and multifunctional 2D material to tailor the potential application of WJ-MSCs in bone tissue regeneration. MSCs can be either applied alone or integrated with scaffolds in the treatment of several diseases, including bone and cartilage dehiscence, wound therapy, and cardiovascular and neural disorders ,2,3,4. M bonding ,22,23.co-glycolic acid) (PLGA) nanofiber [Considering the presence of reactive functional groups at the basal place and edges of GO, researchers have discovered that GO has a great ability to adsorb insulin and dexamethasone through noncovalent interactions ,24,25, wanofiber , hydroxyanofiber , proteinanofiber , and polanofiber  to improa = 5.2 appeared to be negatively charged at a physiological pH of about 7. Thus, the designed peptide should be positively charged at pH 7 for the efficient noncovalent binding of the peptide on the GO film [Synthetic biomaterials, such as peptides and polymers, are easy to fabricate and represent a reliable alternative to in vitro stem cell culture . The uti GO film .In our previous study, we tested the biocompatibility of multilayer GO (m-GO) film toward WJ-MSCs . The preπ–π interactions). Peptide sequences were designed by combining the bioactive short peptide sequences. The short peptide sequences were combined according to the properties of each amino acid, as previously reported by Zhang and coworkers . BrieflyA multilayer graphene oxide (m-GO) film with 25 µg of GO loading prepared according to our previously reported method  was usedThe 3D topography and thickness of both films were analyzed using a Dimension Edge atomic force microscopy  coupled with ScanAsyst-Air cantilevers  at a 0.4 N/m nominal spring constant. The measurement of the film’s surface roughness was calculated using NanoScope Analysis 1.7 software . The functional groups of the fabricated films were examined using Fourier-transform infrared (FTIR) spectroscopy . The surface morphology and composition of both films were measured using an energy dispersive X-ray (EDX) spectroscopy on a scanning electron microscope (SEM) . 2 incubator and replaced with a fresh medium every 2 to 3 days until reaching 80%–90% confluency. When the cells reached confluency, they were detached from the plate by adding 0.125% trypsin–EDTA solution . The cells were then incubated for 3 min at 37 °C incubator.This study was approved by the Ethics and Research Committee of Universiti Malaysia Sabah with approval code JKEtika 1/16 (1). The human Wharton’s jelly mesenchymal stem cells (WJ-MSCs) from the umbilical cord matrix were kindly provided by Dr. Siti Fatimah Simat. The given WJ-MSCs were maintained as described by our previous report . The celn = 3) were sterilized with UV light for 20 min prior to the cellular studies [2 incubator. The medium was changed every 3 days. All the cell assays on the parent m-GO film and peptide/m-GO films were carried out using WJ-MSCs between passage 3 and passage 5. The films , 200 µL of the MTT solution was added to each well and left for 2 h for a cell response to take place. This was followed by the addition of 200 µL of dimethyl sulfoxide to dissolve the purple crystals reduced by the living cells. The optical density of 100 µL of the resulting solution was transferred into new 96-well plate and measured at 570 nm using an Infinite M200Pro microplate reader . The morphology of the WJ-MSCs on the parent m-GO film and peptide/m-GO films was observed using an inverted microscope Olympus IX73 . Prior to viewing by a microscope, the cell medium was discarded, and cells were washed with 1× phosphate-buffered saline . Images of the WJ-MSCs incubated on films under the desired incubation period were captured with either 4×, 10×, or 40× magnification.n = 3) in BM. After 3 days of incubation, the cells were attached. Then, the medium was replaced with the BM or osteogenic medium  according to the manufacturer’s directions. The induction medium was changed every 2–3 days, and the cultures were maintained for the next 14 days. On day 14, the cells were then washed with 1× PBS once and fixed in 300 µL of 10% formalin in PBS for 20 min at room temperature. After fixation, the well plate was washed three times with 1 × PBS and once with distilled water. Osteogenic-induced cells were stained with an Alizarin Red Solution Kit . Prior to observation under an inverted microscope Olympus IX73 , the samples were washed with distilled water three times. The Alizarin Red-stained WJ-MSCs showed maximum expression as the calcium had reached maximum deposition.The parent m-GO film and peptide/m-GO films were placed in a 48-well plate, and WJ-MSCs were seeded at a density of 10,000 cells/well (n = 3) were analyzed using a one-way analysis of variance (ANOVA), followed by a t-test. Results were considered significant when p < 0.05. All the values were presented as ± standard error of the mean (SEM). The numeric data for the MTT assay (The preparation process of the peptide conjugate on the multilayer GO (m-GO) films is shown in −1, respectively. A broad peak at 3395 cm−1 can be ascribed to the O–H vibrations of the hydroxyl group of the GO [−1 as compared with the m-GO film, which indicates that the O–H vibrations of the hydroxyl group on the surface of the m-GO film were diminished in the presence of the peptide. A strong amide I peak (C=O stretching vibrations of the peptide bonds) and a weaker amide II peak (C–N stretching vibrations in combination with N–H bending) were observed at 1650 and 1550 cm−1, respectively. The existence of amide peaks supports the presence of the peptide in all the peptide-conjugated m-GO films [FTIR spectroscopy of the m-GO film and m-GO film conjugated with four different peptide sequences  is shown in f the GO . The FTIGO films ,70. ThesThe presence of carbon (C), oxygen (O), and nitrogen (N) was identified in the parent m-GO film and all peptide-conjugated m-GO films  using energy dispersive X-ray (EDX) spectroscopy . DiffereWe then investigated the potential of our modified m-GO film in biomedical applications. WJ-MSCs were cultured on the m-GO film and peptide-conjugated m-GO films  for up to 6 days. To verify the effect of the peptide on the cell morphology, the cells cultured on samples were observed under bright field microscopy . WJ-MSCsπ–π stacking and electrostatic and hydrogen bonds of the GO/PLL film, resulting in a great enhancement of the osteogenic differentiation of rat-bone-marrow-derived MSCs as compared with GO (GO coverslip) alone [Osteogenesis is one of the major differentiation lineages of MSCs that can be directed by engineering the biophysical properties of biomaterials ,72,73. Tp) alone . Our pepIn conclusion, a new type of peptide/m-GO films was fabricated through mild and inexpensive self-assembly. The fabricated peptide/m-GO films  possessed similar oxygenated groups to the parent m-GO film, with an extra amide peak derived from peptide. The peptide conjugate on the m-GO films promoted the WJ-MSC proliferation and enhanced the osteogenic differentiation based on qualitative analysis. Overall, the peptide/m-GO films could have a promising role in the significant enhancement of controlling the osteogenic lineage of WJ-MSCs for future application in bone regeneration, as well as tissue engineering, due to their easy fabrication process and biocompatibility. However, further studies with quantitative analysis, such as alkaline phosphatase (ALP) activity quantification, calcium content quantification, and gene expression analysis of osteogenic markers by RT-qPCR, are required to further confirm the effects of peptide/m-GO films on WJ-MSCs. In vivo studies are also required to confirm the hypothesis for clinical application.


Mycobacterium bovis BCG Moscow did not induce NK cell activation at 15–20 days post-revaccination. As hypothesized, revaccination with BCG Moscow was associated with a lower incidence of COVID-19 positivity, though the results did not reach statistical significance. Further studies should be carried out to assess whether revaccination with BCG is able to protect HCWs against COVID-19. The protocol of this clinical trial was registered on August 5th, 2020, at REBEC  and the WHO (# U1111-1256-3892). The clinical trial protocol was approved by the Comissão Nacional de ética de pesquisa- CONEP (CAAE 31783720.0.0000.5078).The Bacillus Calmette-Guérin (BCG) vaccine, which is widely used to protect children against tuberculosis, can also improve immune response against viral infections. This unicentric, randomized-controlled clinical trial assessed the efficacy and safety of revaccination with BCG Moscow in reducing the positivity and symptoms of COVID-19 in health care workers (HCWs) during the COVID-19 pandemic. HCWs who had negative COVID-19 IgM and IgG and who dedicated at least eight hours per week in facilities that attended to individuals suspected of having COVID-19 were included in the study and were followed for 7, 15, 30, 60, and 180 days by telemedicine. The HCWs were randomly allocated to a revaccinated with BCG group, which received the BCG vaccine, or an unvaccinated group. Revaccination with BCG Moscow was found to be safe, and its efficacy ranged from 30.0% (95.0%CI -78.0 to 72.0%) to 31.0% (95.0%CI -74.0 to 74.0%).  On March 11, 2020, the World Health Organization (WHO) declared the Coronavirus Disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), as a pandemic . In addiMycobacterium bovis, this vaccine prevents disseminated childhood tuberculosis (TB) and meningitis  vaccine has been safely and widely used in newborns and children for 100 years. Composed of attenuated ningitis . Previouningitis , 8. Otheningitis . Later, ningitis . Shortlyningitis . It has ningitis , 14.The mild manifestations of COVID-19 among children, seen in ecological studies performed in the early stages of the pandemic, were associated with trained immunity generated using vaccines such as BCG . Howeverth, 2020, and August 31st, 2021. The protocol of this clinical trial was registered on August 5th, 2020, at REBEC  and the WHO (# U1111-1256-3892). The clinical trial protocol was approved by the Comissão Nacional de ética em pesquisa- CONEP (CAAE 31783720.0.0000.5078). The detailed study protocol was published as appendix data in a previous publication , Brazil, between August 20lication . This st3 were not enrolled in the study. Participants were randomized to one of two arms: a revaccination with M. bovis BCG Moscow arm or an unvaccinated arm (no placebo vaccine was used). This study was conducted in accordance with the requirements for Good Clinical Practice ICH E6 (R2).The HCWs were recruited at Hospital das Clínicas (HC/UFG) or Hospital Estadual Geral de Goiânia Dr. Alberto Rassi (HGG), or by independently completing an online recruitment form. Inclusion and exclusion criteria were described previously  and are Blood samples from all included HCWs were processed and serum, plasma, PBMCs, and total blood were stored for immunological assays. Initially, enrolled subjects were subsequently excluded if they received a COVID-19-positive test on day 15 after randomization. However, the study protocol was modified and participants who tested positive for COVID-19 within the first 15 days after randomization were kept in the study. This alteration was approved by the Comissão Nacional de Ética em Pesquisa- CONEP and did not affect the integrity of the interpretation of the results obtained and allowed for the assessment of the maximum accurate description of the efficacy of the BCG vaccine since its administration.Flu-like syndrome symptoms according to the recommendations of the Brazilian Ministry of Health were defined as: dry or productive cough, hemoptysis, fever, sore throat, night sweats, shortness of breath, reduced sense of smell or taste, lack of appetite, diarrhea, headache, rhinorrhea, nasal congestion, asthenia/fatigue, and/or myalgia .COVID-19 was considered for an individual presenting an IgM/IgG test or RT-PCR positive test. Any participant that presented symptoms related to flu-like syndrome or symptoms associated to COVID-19 were oriented to perform an RT-PCR test. The severity of COVID-19 was classified according to the WHO guidelines  as follovia intradermal administration. All participants were followed and evaluated through telemedicine on days 7, 15, 30, 60, and 180. An assessment tool designed for this study to evaluate signs of flu-like syndrome symptoms .Adverse events (AEs) and serious adverse events (SAEs) were reported from baseline until the last patient’s evaluation for all included participants. SAEs and all other AEs were reported according to NIH and FDA guidelines , 24. An BCG vaccine-related AEs (adverse reactions) were questioned to all trial participants. As this study did not use a placebo vaccine, only participants allocated to the BCG-vaccinated group reported any vaccine-related AE. Therefore, vaccine-related AEs were measured in analysis only for the intervention group. According to the Brazilian Ministry of Health , severe http://www.jerrydallal.com/random/permute.htm) in blocks of 20  with the permutable treatment labels “BCG vaccination” and “No vaccination” to designate whether the individual would be vaccinated or not, respectively. The total number of subjects was set to 400, and the randomization was divided into blocks of 20 with 20 subjects each. A collaborator who was not involved with the recruitment manipulated the randomization output. Four hundred envelopes externally labeled with the consecutive numbers BCG001 to BCG400 received the respective randomization output printed on a piece of paper with the same BCG number and the result “BCG vaccination” or “No vaccination” generated by the computer software. All 400 envelopes were sealed and ready as of day 1 of the subjects’ recruitment. On the day of recruitment, a consecutive BCG number was assigned to each individual included in the study and a sealed envelope with the exact same BCG number was opened in front of the participant, at which point both the participant and the study staff learned whether the individual was allocated to the vaccination group or not. If allocated to the vaccination group, the individual was transported to the vaccination site; if not, the individual was informed once more about the importance of the unvaccinated group for the trial and their contribution in continuing to participate in the project until the end.Randomization of 400 consecutive numbers was performed on the online platform Randomization.com . Cells were stimulated with medium or with culture filtrate proteins (CFPs) from BCG (0.5 μg/μL) after resting for two hours at 37°C in a 5% CO2 incubator. Then, the cells were incubated for 17 hours, and monensin  was added to the cultures, which were further incubated for 5 hours. After this period, the plates were centrifuged at 2000 xg for 15 minutes at 10°C and the cells were resuspended and treated with 20 μL of mouse sera to block FC receptors. After 10 minutes of incubation at 4–8°C, the cells were incubated for 30 minutes at the same temperature with monoclonal antibodies against surface markers diluted according to a previous standardization. According to the analysis that was to be performed, a combination of the following antibodies were used: mouse anti-human CD16-FITC, CD314-PerCP-eFluor™ 710, TCR-PE-Cyanine7, CD57-eFluor 660, CD27-APC, CD3-Alexa Fluor 700, and CD56 APC-eFluor 780 . All blood samples were kept at -80°C until their cell preparations for cytometry. Using a 37°C water bath, the cells were quickly thawed and distributed in 50 mL tubes or 48-well plates. NK cell staining was performed according to a protocol standardized in our laboratory. In detail, on the day of the cell analyses, two samples of blood from each participant were processed, one collected on the day of recruitment, and one collected at 15–20 days post-randomization. To evaluate NK cell activation, 500 μL of the cell suspensions were transferred to 50 mL conical tubes and erythrocytes were lysed with lysis buffer . After washing the cells with saline, the cell pellet was resuspended with 800 μL of complete RPMI  containing glutamine , pyruvate , non-essential amino acids , 50 μg/mL of penicillin/streptomycin (1.000 U/mL GIBCO) and 10% BSA. The cells were counted and adjusted to 10The sample size calculation, which was already published , was perThe unvaccinated and BCG groups had their demographic, clinical, and behavioral characteristics analyzed and compared. Pearson’s chi-squared test with Yates’ continuity correction or Fisher’s exact tests were used to compare the proportions of categorical variables. A Student’s t-test  or a Mann-Whitney U test with continuity correction  was used to compare the means or distributions of continuous variables. The analysis of the normality of continuous variables was performed using the Kolmogorov-Smirnov test with Lilliefors correction. Parametric quantitative variables were evaluated using a paired t-test or a Wilcoxon test; for non-parametric quantitative variables.P-value <0.10 observed in group comparisons. Also, the log of the risk period to the primary outcome was used as the model’s offset variable to adjust for participants with different study follow-up times. A Cox proportional regression model was used to estimate the hazard ratio (HR). The terms included in this model (explanatory variables) were the study group (BCG group versus control group), age at randomization, and sex. VE was defined as the percentage reduction in the IRR or HR for the primary outcome and was calculated as 1-aIRR (Poisson model) or 1-HR (Cox model), accompanied by the respective 95% CI. Cumulative incidences of cases were presented using the Kaplan-Meier method.Population analysis was performed to estimate VE in the prevention of COVID-19. VE was calculated by two statistical methods: a Poisson regression model and a Cox proportional regression model. A Poisson regression model was used to estimate the incidence rate ratio (IRR) . The terThe first vaccine against COVID-19 was approved for use in Brazil in January 2021, during the follow-up of participants in this clinical trial. Thus, two analyses were performed for VE at endpoints. In the first scenario, entitled “without censoring for COVID-19 vaccination”, we included all cases of COVID-19 until the end of the follow-up, regardless of the participant’s COVID-19 vaccination status. In this case, the time of the event was from the date of inclusion to the date of development of the primary outcome; the participant was censored when (i) follow-up was missed or (ii) 180 days of follow-up had been completed. In the second scenario, entitled “with censoring for COVID-19 vaccination”, individuals were censored from the analysis after 14 days of the subject’s first dose of COVID-19 vaccination, independent of the producing laboratory. In this scenario, the participant was censored when (i) the follow-up was missed (ii) after 14 days of the first vaccine dose (if the participant received the COVID-19 vaccine), or (iii) 180 days of follow-up had been completed (if the participant did not receive the COVID-19 vaccine). Similarly, vaccine efficacies were evaluated according to symptomatic COVID-19 cases.The protocol was registered on August 5th, 2020, at REBEC  and the WHO (# U1111-1256-3892). The clinical trial protocol was approved by the Brazilian Ethics Committee (CAAE 31783720.0.0000.5078).This study complied with guidelines outlined under the Consolidated Standards of Reporting Trials (CONSORT).This clinical trial recruited 592 HCWs, of whom 454 were ineligible  and 138 were randomized, as summarized in P > 0.05). In addition, all HCWs included in the study reported a previous history of BCG vaccination and there was no statistically significant difference between the percentage of individuals with and without a scar from previous BCG vaccination between the BCG-revaccinated and unvaccinated groups.The baseline characteristics of participants according to allocated group (unvaccinated and revaccinated with BCG) are shown in As detailed in methods, during the trial period, specific COVID-19 vaccines became available to HCWs, thus the VE is presented in two scenarios Table 3.The Kaplan-Meier cumulative incidences of symptomatic COVID-19 were evaluated for the scenarios without censoring for COVID-19 vaccination Table 4.Additionally, the efficacies calculated considering only HCWs that presented previous BCG scar showed no significant differences between the groups that were revaccinated with BCG or unvaccinated .The comparison of general symptoms reported by HCWs included in the unvaccinated and revaccinated with BCG groups is described in The SARS-CoV-2 exposure profile and risk of COVID-19 infection among included HCWs who had COVID-19 according to their occupation is described in The local reactogenicity of the BCG vaccine was evaluated for all groups, revaccinated or unvaccinated. The HCWs revaccinated with BCG reported some vaccine-related AEs. However, as expected, the non-revaccinated group, for which no placebo was used, did not report BCG vaccine-related AEs. Of the total number of individuals (64), sixty-three (98.4%) had local adverse events related to BCG, the three most frequent of which were erythema , papule , and crust . The median number of local AEs related to BCG vaccination was five (IQR=4–6) Table 5.P > 0.05). The immune response results presented high variability; thus, the fold of increase of these cell populations was evaluated, comparing day 15 relative to day 1 post-randomization. As shown, no differences were observed when the fold of increase of activated cells was evaluated (P > 0.05).To verify the responsiveness of NK IFN-γ- or TNF-α-positive cells, total blood was stimulated with BCG Moscow culture filtrate proteins (CFPs). It was possible to observe that the unvaccinated and revaccinated with BCG groups had similar levels of NK IFN-γ- or TNF-α-positive cells, on both day 1 and day 15 (P < 0.05), indicating a possible role for NK cells that requires further study. No differences were observed when analyzing the other symptoms. Furthermore, the fold of increase analysis of NK TNF-α-positive cells showed no differences between symptomatic and non-symptomatic individuals (P > 0.05).BCG vaccinations were hypothesized as inductors of NK responses that could reduce SARS-COV-2 cell propagation and therefore reduce COVID-19 symptoms. Although no differences were observed in the evaluated immune responses, it was questioned whether previously activated NK cells could have an impact on the symptoms associated with COVID-19. Participants who had a higher fold of increase of NK IFN-γ in the 15–20 days after randomization presented fewer incidences of fever and dyspnea (P < 0.05). No differences were observed when the fold of increase of NK TNF-α-positive cells was evaluated (P > 0.05).The positivity for COVID-19 or the development of flu-like syndrome symptoms during the trial was analyzed with the fold of increase of NK IFN-γ cells. It was observed that COVID-19-positive individuals or those that presented flu-like symptoms had a lower fold of increase of NK IFN-γ-positive cells when compared to COVID-19-negative individuals or those that presented flu-like syndrome, respectively  suggested that the BCG vaccine did not protect vulnerable elderly people from COVID-19. Although the study included and monitored more than 6,132 sixty-year-old individuals for 180 days after BCG vaccination, the results revealed that COVID-19 infection in combination with disease symptoms occurred with the same frequency in the elderly from the control group and those vaccinated with BCG. However, it is important to note that almost all individuals participating in the BCG-PRIME study received the BCG vaccine for the first time, as the Netherlands did not adopt a policy of vaccination with BCG in childhood .In contrast, the double-blind, randomized trial (ACTIVATE-2) performed with Greek volunteers demonstrated that BCG revaccination resulted in a 68% risk reduction for total COVID19 diagnoses (OR 0.32. 95% CI 0.13-0.79). However, in that study, the diagnosis of COVID-19 of some participants was based on the clinical diagnosis of possible or probable COVID-19 . AnotherAdditionally, study carried out by Rivas et al.  demonstrMycobacterium tuberculosis (Mtb) resulted in an increase in IFN-γ specific to Mtb at 90 days but is not observable at 14 days after vaccination. Stimulation with non-specific components results in the production of IL-6 at 14 and 90 days after vaccination . This vaccine contributes to an increase in plasma levels of IFN-γ and IL-10 after six months of administration . More recination . In a stcination . Howevere months , 54. HowIt is known that the protection conferred by BCG against tuberculosis depends on the induction of a specific response, Th1 ; howeverIL-6 and TNF genes and increased TNF-α, IL-1β, and IL-10 cytokine synthesis after three months of immunization. Apparently, the BCG Bulgaria vaccine induces an effective heterologous response, which may have contributed to a cross-response against other infections. Different from the results observed by Giamarellos-Bourboulis et al. (+CD56dim) cells after 14 days of immunization, which did not enhance their production of IFN-γ or TNF-α. Our work, although employing a different methodology, presented similar results as those observed by Kleinnijenhuis et al. , Ministério da Ciência, Tecnologia e Inovações (MCTI)/Ministry of Science, Technology and Innovations, Grant numbers: . Furthermore, the BCG vaccines were kindly donated by the Brazilian Immunization Program of the Health Ministry of the Brazilian Government/Programa Nacional de Imunização, Ministério da Saúde, Brasil. Neither the funding agency nor the Brazilian Immunization Program had a role in the trial design, the conception, collection, analysis, interpretation, or conclusions, or the decision to write this manuscript. LRBA received fellowship from Coordenação de Pessoal de nível Superior (CAPES-Finance Code 001) and KMR, CCS, ACOC, and AOR received fellowship from CNPq.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Bladder cancer (BCa) and prostate cancer (PCa) are some of the most common cancers in the world. In both BCa and PCa, the diagnosis is often confirmed with an invasive technique that carries a risk to the patient. Consequently, a non-invasive diagnostic approach would be medically desirable and beneficial to the patient. The use of volatile organic compounds (VOCs) for disease diagnosis, including cancer, is a promising research area that could support the diagnosis process. In this study, we investigated the urinary VOC profiles in BCa, PCa patients and non-cancerous controls by using gas chromatography-ion mobility spectrometry (GC-IMS) and gas chromatography time-of-flight mass spectrometry (GC-TOF-MS) to analyse patient samples. GC-IMS separated BCa from PCa (area under the curve: AUC: 0.97 (0.93–1.00)), BCa vs. non-cancerous (AUC: 0.95 (0.90–0.99)) and PCa vs. non-cancerous (AUC: 0.89 (0.83–0.94)) whereas GC-TOF-MS differentiated BCa from PCa (AUC: 0.84 (0.73–0.93)), BCa vs. non-cancerous (AUC: 0.81 (0.70–0.90)) and PCa vs. non-cancerous (AUC: 0.94 (0.90–0.97)). According to our study, a total of 34 biomarkers were found using GC-TOF-MS data, of which 13 VOCs were associated with BCa, seven were associated with PCa, and 14 VOCs were found in the comparison of BCa and PCa. Early detection and diagnosis of cancer remains a key goal to improve the prognosis and life expectancy of patients ,2,3,4. GBladder cancer (BCa) is the ninth most common cancer worldwide and is also one of the most difficult cancers to diagnose and clinically manage ,9. CystoProstate cancer (PCa) occurs in men and is the sixth most common cancer worldwide ,15,16. FOne area receiving significant interest is in the use of volatile organic compounds (VOCs) to diagnose and monitor cancer. VOCs are chemical compounds that are either produced in vitro or are introduced externally and can indicate the presence, or absence, of disease in the body. The concept first emerged after reports indicated that dogs could recognise cancer by sniffing biological samples . Since tUrine is a common biological source of VOCs, as the components present are either the intermediate products or end products of metabolic activities occurring inside the human body . A studyUrine cytology is a non-invasive test which uses urine as biological modality for the presence of cancer. Several studies showed that though it exhibits high specificity, the sensitivity and specificity highly depend on collection method and cancer grade ,31,32.The gold standard for the analysis of VOCs remains GC-MS, but it is expensive, requiring specialised equipment and trained staff, making it difficult to implement in a point of care scenario. A variant of this is GC-TOF-MS (gas chromatography-time of flight-mass spectrometry), which is a similar technique used for multidimensional analysis of complex samples with the potential to identify an even greater number of VOCs ,34. HoweThe study aimed to identify and test the potential of urinary biomarkers to distinguish between two different cancers and healthy controls using both GC-TOF-MS and GC-IMS. We believe this is the first time that GC-IMS has been used with these cancers in combination with GC-TOF-MS.A total of 106 patients were recruited after providing informed consent at University Hospital Coventry and Warwickshire NHS Trust, UK. Patients were recruited prior to anti-cancer treatment. This study was approved by Coventry and Warwickshire and North-East Yorkshire NHS Ethics Committees (Ref 18717 and Ref 260179). Urine samples were collected in standard universal sterile specimen containers and frozen within 2 h at −80 °C for subsequent batch analysis and according to standard operating procedures, compliant with tissue bank requirements under Human Tissue Act 2004. No chemicals were added to the urine before freezing, as we have previously shown that urine samples remain stable for extended periods of time at this temperature . Prior tThe G.A.S FlavourSpec (Germany) uses a GC-IMS measurement technique to analyse VOCs. GC-IMS is a method used in various applications, such as detection of explosives and chemicals ,41,42, aFor analysis, glass vials containing samples were transferred to an autosampler fitted to the GC-IMS. The sample tray was chilled to 4 °C to reduce sample degradation during sample analysis. Each sample was heated to 40 °C and agitated for 10 min before sampling. The autosampler then took 0.5 mL of sample headspace and directly injected it into the GC-IMS. Urinary headspace was defined as the volume of gas above the urine sample inside the vial, which was in chemical equilibrium with liquid phase urine. The machine settings for analysis were as follows: E1: 150 mL/min (for the drift tube IMS), E2: 20 mL/min (for the GC column), and the pump was set to 25%. The total run time per sample was 10 min. The temperatures were set to T1 (IMS): 45 °C, T2 (column): 80 °C, and T3 (injector): 70 °C.GC-TOF-MS operates by analysing the time of flight of ions and analyse them according to their mass-to-charge ratio. The GC-TOF-MS system used was a combination of a TRACE 1300 GC  and a BenchTOF-HD TOF-MS . This system also included a high-throughput autosampler and a thermal desorption unit, ULTRA-xr and UNITY-xr, respectively (both from Markes Intl.). The GC separated the chemicals in the same way as explained previously. The separated chemicals were detected by TOF MS once they entered the TOF ‘flight box’. TOF-MS separates fragment ions instead of molecular ions as in an IMS. The ions are detected depending upon the mass-to-charge ratio of the ions after passing through the drift tube ,53.For analysis, a thermal desorption (TD) sorbent tube  was inserted through the septum and into the headspace above the sample and then heated at 40 °C for 20 min. A pump was then attached to the TD tube, and whilst still being heated to 40 °C, the headspace VOCs were then pulled onto the tubes at 20 mL/minute for a further 20 min. The sorbent tubes were then placed in an autosampler for analysis. The analysis started with ULTRA-xr with a stand-by split set to 150 °C. The GC run time for samples was 25 min with a programmed temperature ramp from 40 °C to 280 °C at 20 °C/min. Each sample was pre-purged for 1 min and then desorbed at 250 °C for 10 min, with the trap purge time set to 1 min. These traps were then cooled at −30 °C and the trap was purged for 3 min at a temperature of 300 °C. The temperature for both transfer line and ion source were heated to 250 °C. The chemicals from GC-TOF-MS analysis were identified using the national institute of standards and technology (NIST) list (2011).For GC-IMS data analysis, the data were extracted using the G.A.S VOCal  software. This was followed by pre-processing steps to reduce the data’s dimensionality. Among all the data points, the central section contained all the computationally significant chemical information and thus all the other data were removed through a cropping process. This was followed by applying a small threshold to remove the background information, which was a value just above the background noise level. The same data cropping and threshold values were used on all the data, and it was undertaken using an automated program. The data were then analysed using a 10-fold cross-validation, undertaken using a bespoke R program (version 3.6.2). Within each fold training set, feature selection was undertaken using a Wilcoxon rank-sum test between the different cancer groups and non-cancerous group. That resulted in the identification of the 20 most discriminatory features between the two groups and the features trained by three models, XGBoost, logistic regression, and random forest. The model was then applied to the test set to create class probabilities. Once all the samples had been within a test set, statistical results were generated from the probabilities, including a receiver operator characteristic (ROC) curve, area under the curve (AUC), sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV).An analogous approach was used for GC-TOF-MS data analysis. For GC-TOF-MS, the chemicals and the abundance of the chemicals were identified. Using the TOF-DS software, a background correction was applied, and the chromatogram was integrated, and the peaks were identified using the NIST list which was exported. The data obtained from GC-TOF-MS were converted into text files of chemical lists and abundances. The data were then processed using an ‘R’ program that was like that used for GC-IMS, where chemical components of discriminative power were identified. x-axis represents the drift time of the IMS and the y-axis represents the retention time of the GC. In the figure, the ‘dots’ are the chemicals detected by the IMS and the intensity of the peak represents the number of ions. Those ‘dots’ in red are the most intense. The red line in the figure is the default output of the instrument where no chemicals are present. G.A.S VOCal  was used to view the GC-IMS data.x-axis refers to the retention time, and the y-axis, the total ion count.The results of the statistical analysis of the GC-IMS gathered results between different cancer groups and the non-cancerous group are given in The ROC curves obtained from GC-IMS data comparing BCa and the non-cancerous group, BCa and PCa groups, and PCa and non-cancerous groups are shown in The results of the statistical analysis between different cancer groups for GC-TOF-IMS are given in The ROC curves obtained from GC-TOF-MS data comparing BCa and non-cancerous groups, BCa and PCa groups, and PCa and non-cancerous groups are shown in In our results, we analysed different VOCs linked to BCa and PCa for the screening and diagnosis of these cancers. A total of 34 biomarkers were found using TOF-DS software. These VOCs were verified using PubChem, NIST , and previously published papers. Out of 34, 13 VOCs were found in the comparison of BCa and non-cancerous groups specific to BCa, as shown in In our study, we found that both GC-IMS and GC-TOF-MS were able to separate different cancer groups from each other as well as non-cancerous group. The separation between BCa from non-cancerous group was highest using GC-IMS with 0.95 AUC (0.87 sensitivity and 0.92 specificity). A similar study conducted by Weber et al.  suggesteThe separation between PCa and the non-cancerous group was highest using GC-TOF-MS method with 0.94 AUC (0.78 sensitivity and 0.88 specificity) whereas the study conducted by Gao et al.  for the In this study, we developed urinary VOC profiles linked with BCa and PCa. Out of 13 VOCs found to be noteworthy to BCa, biphenyl, heptanal, and 2,6,10,14-tetramethyl-pentadecane were the three distinct biomarkers found in our study that did not overlap with other studies. Biphenyl has been identified as the most significant biomarker in our study. Biphenyl has been linked to various diseases, including carcinoma. It has been proven that biphenyl is a promoter of BCa in rats . BiphenyFrom the analysis of PCa urine samples, a total of seven distinct VOCs were identified and are summarised in The use of urinary VOC analysis is an attractive option due to the non-invasive nature. It also has the potential to be used in early cancer diagnosis with further validation studies. This approach may also prove to be efficient, whilst lowering the cost per patient, and increasing patient compliance due to its non-invasive nature. The results of using GC-IMS as an analysis tool are significant as the method is much simpler than using a high-end analytical method, such as GC-MS, and without the need for a laboratory environment. We believe that using VOCs to analyse human waste will be an important diagnostic tool for the future. Cancer may well be one area of focus and may be used as part of the UK 2-week wait screening program to help reduce the number of unneeded procedures. The key is to run more larger studies targeting these cancers and to have tools that are CE marked  for cancer diagnosis. We plan to use urine VOCs in association with other tests in future which help to improve the performance and achieve a more in depth understanding of VOCs and their metabolic pathways.Our results were limited by not accounting for the contributory factors that can also lead to abnormal metabolism with subsequent excretion of differing concentrations of these chemicals in the urine. These factors include stress, alcohol, smoking, certain food products, medicines, and different environmental factors. Several studies have reported the effect of smoking on VOCs ,75. StudIn this paper, GC-IMS and GC-TOF-MS methods were used to identify VOC fingerprints using urine headspace and establish an interdependence between BCa, PCa and non-cancerous samples. It was found that both GC-IMS and GC-TOF-MS have the potential to differentiate between different cancer groups with respective AUC for different diagnostic groups: For GC-IMS, BCa and PCa (0.97 (0.93–1)), BCa and non-cancerous (0.95 (0.90–0.99)), PCa and non-cancerous (0.89 (0.83–0.94)) and for GC-TOF-MS, BCa and PCa (0.84 (0.73–0.93)), BCa and non-cancerous (0.81(0.70–0.90)), PCa and non-cancerous (0.94 (0.90–0.97)). A total of 35 VOCs were found to be relevant for identifying these cancer groups, with several VOCs distinct to each cancer. VOCs from this study were supported by findings from previous studies. This signifies that VOCs for both bladder and prostate cancer have different profiles, which may be helpful in future to distinguish them. In the future, the VOC profiles obtained from these analytical devices can be used as a reference for developing low-cost devices. It is plausible that VOC profiles can be used as an adjunct to diagnosis enabling selection of only high-risk groups to undergo cystoscopy examinations which will be widely beneficial considering limited capacity and cost.


Aspherical- and multi-curve rigid gas-permeable hard contact lenses (HCLs) have a flattened curve in the peripheral zone and are mostly used for patients with keratoconus who cannot wear glasses, soft contact lenses, or spherical HCLs. In this retrospective study, a total of 95 eyes of 77 patients who used aspherical- or multi-curve HCLs (mean age: 40.0 ± 11.0 years) were evaluated. This study examined the types of aspherical- and multi-curve HCLs, best-corrected visual acuity (BCVA) values before and after wearing HCLs, the association with the Amsler-Krumeich classification, duration of wear, corneal/conjunctival disorder, and the frequency of changing HCLs. There were 78 eyes that used aspherical-curve HCLs and 17 that used multi-curve HCLs. BCVA significantly improved from 0.42 logMAR to 0.06 logMAR after wearing either form of HCL. The Amsler-Krumeich classification showed that aspherical-curve HCLs were commonly used for patients with stage 2 keratoconus, and multi-curve HCLs were commonly used for stage 4 patients. The BCVA values were worse when the disease stage was more severe (stages 3 and 4) regardless of HCL type. The mean base curve of the lenses was steeper in multi-curve HCLs than in aspherical-curve HCLs. The more severe the disease stage, the steeper the base curve in both aspherical- and multi-curve HCLs. The duration of wear significantly improved from 2.1 h to 10.2 h, and corneal/conjunctival disorder similarly improved. The mean frequency of changing HCL types was 1.1 times. This study suggests that a flat peripheral curve design with aspherical- and multi-curve HCLs is useful for patients with keratoconus. Keratoconus is a bilateral corneal dysmorphic disorder in which the center of the corneal stroma is thinned and protrudes anteriorly. The changes in corneal shape are accompanied by abnormalities in visual function. The cause of the disease remains unknown, despite various reports –3. The iThe normal corneal structure has an elliptical shape that mildly steepens in the central corneal zone and is almost uniformly flat between the intermediate corneal zone and the peripheral corneal zone. The value of the radius of corneal curvature, the index of the corneal shape, changes evenly from the central corneal zone through the peripheral corneal zone. In eyes with keratoconus, the corneal apex can be in the lower region rather than in the central region, and the central cornel apex is severely protruded. Consequently, the corneal shape may change unevenly. In early-stage keratoconus, the corneal shape is close to normal, and the degree of astigmatism is small.Visual correction for early-stage keratoconus is possible with glasses and soft contact lenses (SCLs); however, rigid gas-permeable hard contact lenses (HCLs) are usually indicated . Among sAspherical- and multi-curve HCLs are designed with a radius of curvature in the peripheral zone that is non-uniformly prepared relative to the BC. These HCLs can correct the vision even in advanced keratoconus –9. HCLs This study aimed to retrospectively assess the outcomes of treatment with aspherical- or spherical multi-curve HCLs  with flat curves in the peripheral zone for patients with keratoconus for whom vision could not be corrected with glasses, SCLs, or spherical HCLs.The study was approved by the Institutional Review Board of Kitasato University Hospital (B19-365) and was performed in accordance with the Declaration of Helsinki. The Institutional Review Board waived the requirement for informed consent for the review of the clinical charts.The study comprised a total of 95 eyes of 77 consecutive patients with keratoconus  who were diagnosed at Kitasato University Hospital between January 2016 and December 2018. The overall follow-up period was 19.7 ± 7.9 months, with mean follow-up periods for aspherical- and multi-curve HCLs of 17.5 ± 8.8 and 21.0 ± 7.2 months, respectively.The indication criteria were patients who could not wear glasses, SCLs, or spherical HCLs. In contrast, exclusion criteria were inflammatory eye disease, glaucoma, cataract, retinal disease, or giant papillary conjunctivitis.For the diagnosis of keratoconus, the corneal shape was measured using corneal anterior segment optical coherence tomography (AS-OCT) . The data are presented using an absolute scale. Clinical findings, such as protrusion and thinning of the corneal apex, Vogt’s striae, and Fleischer’s ring, were observed using a slit-lamp microscope .For data measurement, the best-corrected visual acuity (BCVA) in the logarithm of the minimum angle of resolution (logMAR) was measured before and after wearing aspherical- or multi-curve HCLs. The outcomes of wearing aspherical- and multi-curve HCLs were analyzed. Subsequently, we analyzed the association of the severity of keratoconus with the BCVA values and the outcomes of wearing HCLs. Keratoconus severity was classified using the Amsler-Krumeich classification system . The mea®) and multi-curve HCLs (HI SANSOα-Multi-curve lens®) used in this study were obtained from Rainbow Optical Contact Lens Corporation . Aspherical-curve HCLs have an optical zone with a spherical BC and a peripheral zone with an aspheric structure designed using a conchoid curve. The constant that determines the shape of a curve is defined as eccentricity (E); the larger the E value, the flatter the peripheral zone curve. We designed three types of aspherical-curve HCLs characterized by an E value of 3, 4, or 5 of the peripheral zone curve  , Table 1HCL parameter selection, the radius of corneal curvature as measured by AS-OCT, and BCs of HCLs were compared to select trial lenses. After the trial lenses were worn, fluorescein patterns of HCL staining, fitting, centering, and movement using slit-lamp microscopy were assessed. HCL fitting on the cornea was performed based on a three-point-touch technique to determine the best HCL.Statistical analyses were performed using an add-in software for Microsoft Excel . The normality of all data samples was first checked using the chi-squared distribution test. Since none of the data were normally distributed, the data were compared using the Wilcoxon signed-rank test and Mann–Whitney U test. The stages 1 to 4 keratoconus data were compared using one-way analysis of variance (ANOVA) when possible. The data are presented as means ± standard deviation, and a value of p <0.05 was considered statistically significant.Of the 95 eyes, 78  wore aspherical-curve HCLs, and 17  wore multi-curve HCLs .The BCVA significantly improved from 0.42 ± 0.45 to 0.06 ± 0.16 logMAR in the whole study population . The BCVA in the aspherical-curve HCL group improved from 0.37 ± 0.42 to 0.06 ± 0.13 logMAR, and those in the multi-curve HCL group improved from 0.66 ± 0.55 to 0.05 ± 0.06 logMAR. There was no significant difference in the BCVA between aspherical- and multi-curve HCL groups .The overall BC was 6.59 ± 0.6 mm in the whole study population. The BC in the aspherical-curve HCL group was 7.20 ± 0.5 mm, and that in the multi-curve HCL group was 6.13 ± 0.4 mm .According to the Amsler-Krumeich classification, the BCVA was worse in more severe stages (stages 3 and 4) than in mild stages (stages 1 and 2) in both aspherical- and multi-curve HCL groups, with a significant difference observed  .The E4 type lenses were mostly used in the aspherical-curve HCL group, and the 4-level steep-type lenses were mostly used in the multi-curve HCL group .Aspherical-curve HCLs were used in 13 (17%) eyes with stage 1 keratoconus, 30 (38%) with stage 2, 12 (15%) eyes with stage 3, and 18 (23%) eyes with stage 4. Multi-curve HCLs were used in 3 (18%) eyes with stage 2 keratoconus, 1 (6%) eye with stage 3, 10 (59%) eyes with stage 4, and none for stage 1 (0%).The more severe the stage, the steeper the BC was in eyes that used both aspherical- and multi-curve HCLs . The mean BC in the multi-curve HCL group was steeper than that in the aspherical-curve HCL group, especially in stages 2 and 4 .The duration of HCL wear significantly increased from 2.1 ± 4.2 h to 10.1 ± 3.7 h in the entire study population  . The totAt the final follow-up, we found significant differences in mean keratometry  and minimum corneal thickness (p = 0.005) between the two groups; however, no significant differences in corneal astigmatism or central corneal thickness were observed .In this study, we designed aspherical- and multi-curve HCLs with flat peripheral zones to treat keratoconus. Our findings indicate that a flat peripheral curve design with aspherical- and multi-curve HCLs is clinically beneficial, especially for patients with advanced keratoconus, resulting in a decrease in the number of surgical cases for penetrating keratoplasty or deep anterior lamellar keratoplasty. We used the AS-OCT for the diagnosis and the grade classification of keratoconus. Maeda et al.  and NadeThe current findings demonstrated a significant BCVA improvement during wearing aspherical- and multi-curve HCLs. The mean durations of HCL wear were 10.2 ± 3.7 h for aspherical-curve HCLs and 11.3 ± 3.1 h for multi-curve HCLs ; howeverAspherical-curve HCLs were often used for patients in keratoconus stages 2 and 3, whereas multi-curve HCLs were often used for those in stage 4. We assume that this is because as keratoconus progresses, the corneal cone protrusion worsens. When aspherical-curve HCL is fitted to a protruded corneal cone, there can be a space between the HCL and the cornea, resulting in a poor HCL fit. On the contrary, even when the corneal cone protrusion worsens, there is less space between the multi-curve HCL and the cornea, resulting in a good HCL fit since the peripheral zone has a multi-level curve. Therefore, it is reasonable that multi-curve HCLs are more suitable for advanced keratoconus.The BC of both aspherical- and multi-curve HCLs steepens in severe stages due to the anterior protrusion of the cornea. The steeper BC of the multi-curve HCLs compared to aspherical-curve HCLs, even in the same stage, may be associated with the position of the corneal cone. We suggest that the BC in the central zone be flat when a corneal cone is present in the lower region, whereas the BC should be steep when the cone is in the central zone .Previous studies on aspherical-curve HCLs have been reported by Yanai et al.  and KazaIn previous studies on multi-curve HCLs, the HCLs were not designed to have a flat peripheral zone curve; however, the BCVA, duration of HCL wear, and HCL wearing success rates were better than those obtained in this study –19. The Higher-order aberrations and contrast sensitivity function were evaluated during HCL wear in some previous studies. Yanai et al.  reportedRegarding vision correction for keratoconus, not only HCLs, but also asymmetric SCLs , scleralKamiya et al.  reportedThere are at least two limitations to this study. Firstly, this study was performed at a single center, and, thus, the sample size was relatively small. Secondly, it is still difficult to objectively evaluate the optimal means of HCL wearing exclusively based on the clinical appearances. Elbendary et al.  measuredA flat peripheral curve design with either aspherical- or multi-curve HCLs was clinically beneficial for patients with keratoconus. Multi-curve HCLs tended to be applied, especially in advanced keratoconus. Aspherical- and multi-curve HCLs may help obtain good visual outcomes for keratoconus without surgical interventions. Nevertheless, a further prospective comparative study with a large cohort of patients with keratoconus is necessary to confirm our findings.


V Flow is commercially developed by high-frame-rate ultrasound vector flow imaging. Compared to conventional color Doppler, V Flow is angle-independent and is capable of measuring both the magnitude and the direction of blood flow velocities. This paper aims to investigate the differences between V Flow and pulsed wave Doppler (PW) relative to phase contrast magnetic resonance imaging (PC-MRI), for the quantitative measurements of blood flow in common carotid arteries (CCA) and, consequently, to evaluate the accuracy of the new technique, V Flow. Sixty-four CCAs were measured using V Flow, PW, and PC-MRI. The maximum velocities, time-averaged mean (TAMEAN) velocities, and volume flow were measured using different imaging technologies. The mean error with standard deviation (Std), the median of absolute errors, and the r-values between V Flow and PC-MRI results for the maximum velocity, the TAMEAN velocity, and the volume flow measurements are {9.40% ± 14.91%; 11.84%; 0.84}, {21.52% ± 14.46%; 19.28%; 0.86}, and {−2.80% ± 14.01%; 10.38%; 0.7}, respectively, and are {53.44% ± 29.68%; 49.79%; 0.74}, {27.83% ± 31.60%; 23.83; 0.71}, and {21.01% ± 29.64%; 25.48%; 0.34}, respectively, for those between PW and PC-MRI. The boxplot, linear regression and Bland–Altman plots were performed for each comparison, which illustrated that the results measured via V Flow rather than via PW agreed more closely with those measured via PC-MRI. Doppler ultrasound is one of the most commonly used medical imaging techniques for the routine examination of vascular disease. In clinics, carotid artery stenosis can be diagnosed using ultrasonography (US) via B-mode grayscale images and the peak systolic (PS) and end-diastolic (ED) flow velocities, which are the maximum values at the location , identified visually using color Doppler, and measured quantitatively by pulsed wave Doppler (PW). The degree of stenosis for carotid arteries can be detected and quantified by measuring the narrowest diameter of the remaining stenotic lumen directly and by comparing it to the distal normal lumen (NASCET ) or the It is essential to make an accurate estimation of blood flow velocities; however, the conventional Doppler US  is angle-dependent, measuring only one-dimensional velocity components along the ultrasound beam, and the actual flow angle has to be corrected according to the shape of the blood vessels, assuming that the blood travels parallel to the vessel wall . This asCompared to conventional Doppler US, vector flow imaging (VFI) is an innovative technology , in whicFor the clinical application of quantitative results-based diagnosis, both the accuracy and reproducibility of the VFI-based measurements must be prioritized. Previously, TO-based VFI has been developed for industry, with measurements validated by magnetic resonance angiography (MRA) ,22. In tThe rest of this paper is structured as follows. This study was approved by the local Institutional Review Board of Tsinghua University. Written informed consent was obtained from all subjects. Two ultrasound techniques and PC-MRI were used for the examination of both the left and right common carotid arteries (CCAs) for all presumed healthy volunteers, including 15 males and 17 females, with an average age of 49, ranging from 27 to 65 years old. Thus, 64 CCAs were measured using the three imaging techniques. Due to the wide age range, it was possible to investigate a wide range of flow velocities for the comparison studies. For each participant, the total examination, including PW, V Flow, and PC-MRI, was completed within a 2 h period on the same day . The ultrasound and MRI scans were performed in the supine position and were operated mainly by a sonographer–L.D. (10-years+ experience with ultrasound systems), and an MR operator–L.H. (20-years+ experience with MRI systems), respectively.V Flow is a high-frame-rate FR: 374~1240 Hz) dynamic displayed ultrasound vector flow imaging technique that is commercially available for the Resona 7 ultrasound system. It employs multi-directional ultrasound transmission and reception to generate velocity components along different angles ,23,25. E0 Hz dynaFor ultrasound scans, the quantitative blood flow measurements for the common carotid artery (CCA) were made using a linear array transducer (L11-3U) via PW and V Flow using a commercial ultrasound system, Resona 7, manufactured by Shenzhen Mindray Bio-Medical Electronics Co., Ltd. . Both PW and V Flow are commercially released functions on the Resona 7 system. The blood flow velocity and volume flow of CCA were measured via PW and V Flow. For PW, the results were corrected using a conventional angle correction line, as shown in the example in For V Flow, flow data were acquired continuously for 1.5 s. The frame rate was around 500~600 fps set by users depending on the estimated velocities. The maximum and time-averaged mean velocities (T-Max and TAMean for V Flow) within a long ROI spanning the vessel diameter  as readout with flip angle—10°; and TR/TE—13.0/7.9 ms. The phase contrast flow direction was the foot–head direction with a velocity encoding (VENC) of 90 cm/s. The peripheral pulse unit (PPU) was used to synchronize the scan with 15 heart phases. The maximum and time-averaged mean velocities and the volume flow rate are estimated for both the left and right common carotid arteries . One example is shown in All MR experiments were performed on a multi-transmit 3T MR scanner  using a 20-channel head–neck coil. The general acquisition parameters for 2D PC-MRI of each CCA were: FOV—150 by 150 mmThe PC-MRI measurements were used as the reference (benchmark). The data were processed for comparison studies by Matlab , which was used to calculate all statistical results and to generate plots. The results were obtained using PW and V Flow for the maximum and TAMEAN velocities, and the volume flows were compared to those obtained using PC-MRI. The mean error with standard deviation and the median of absolute errors were calculated. The boxplot was performed based on the errors relative to PC-MRI results for PW and V Flow. The Pearson correlation coefficient  was calculated for each comparison to study the correlation and similarity of the results measured via different imaging techniques. The linear regression and Bland–Altman plots were also used to illustrate the differences for each comparison.p-value = 0.07  for the volume flow between V Flow and PC-MRI measurements, and p-value < 0.001 for the rest of the compared results. The linear regression and Bland–Altman plots are shown in The relative errors and r-values of V Flow and PW compared to the PC-MRI measurements are listed in p-values < 0.001) can be found in this comparison study. From the boxplot, regression, and Bland–Altman plots, it can be seen that the velocity results are clearly overestimated by PW relative to PC-MRI. For V Flow, the limits of agreement contain the zero line, which means the bias is not statistically different from zero . The spreads of the V Flow vs. PC-MRI measurements are clearly smaller than those of PW vs. PC-MRI, which also have more data points outside the limits of agreement  and overestimation (For the volume flow measurements, different compared results are shown for PW and V Flow. Significant differences (timation  can stiltimation .In previous studies, the TO-based VFI emerged with a higher precision of velocity measurement than PW at different examination sites . As a hiOverall, the estimating results of V Flow, rather than PW, have a better agreement with the PC-MRI measurements. The velocities are overestimated by both PW and V Flow compared to PC-MRI estimations, but with no systemic bias for V Flow. The volume flow is overestimated by PW, but with no significant difference for the volume flow of the V Flow estimation compared to PC-MRI. High-frame-rate vector flow imaging based on multi-directional Doppler transmission and reception is a promising technology that achieves more accurate quantitative measurements for the CCA compared to conventional PW.


Objective: Multiple system atrophy (MSA) is a serious neurodegenerative disease that is charactered by progressive neurological disability. The aim of this study was to investigate the correlation of serum oxidant factors with the severity of MSA.Methods: A total of 52 MSA patients and 52 age- and gender- matched healthy subjects were retrospectively enrolled in this study. Enzymatic colorimetric methods were used to assay the concentrations of uric acid (UA), serum creatinine (Scr), blood urea nitrogen (BUN), and cystatin C (Cys-C). Disease severity was evaluated by the Unified Multiple System Atrophy Rating Scale (UMSARS). The disease progression rate was defined by the change in UMSARS-IV  over a 1-year period.Results: Comparisons between the two groups revealed that there were no significant differences in terms of serum Scr . However, the serum levels of the other three biomarkers were significantly higher in the MSA patients . In addition, Pearson correlation analyses revealed that only serum Cys-C was significantly correlated to GDS . Subgroup analysis further demonstrated that serum Cys-C was the only factor that was positively associated with the disease severity in patients with MSA and predominant cerebellar ataxia (MSA-C) ; there was no significant association in MSA patients with predominant Parkinsonism (MSA-P) . MSA-C patients with severe disability were shown to express higher serum levels of Cys-C than patients with mild disability . Finally, Kaplan-Meier plots revealed a significant difference in the 5-year probability of survival from severe disability between MSA-C patients with high- and low-concentrations of serum Cys-C . ROC curve analysis confirmed that serum Cys-C exhibits good performance as a biomarker (AUC = 0.847).Conclusion: Our research indicated that oxidative stress plays a vital role in MSA. Serum Cys-C represents a potential prognostic biomarker to evaluate the severity of disease in patients with MSA-C. Multiple system atrophy (MSA) is an adult-onset, sporadic, and rapidly progressive neurodegenerative disease that is characterized by autonomic failure in connection with Parkinsonism and/or cerebellar ataxia . The genA significant body of evidence now indicates that oxidative stress plays a key role in triggering and/or exacerbating MSA . OxidatiThis study was a case-control study that took place between June 2013 and December 2018 and involved Chinese patients with MSA. A total of 91 Chinese patients previously diagnosed with MSA were retrospectively identified via diagnosis-specific code (ICD-10 code G90.301). Patients were eligible for inclusion in this study if they met the following criteria: (1) if diagnosis had been made on the basis of the second consensus criteria , (2) theSerum levels of UA, Scr, BUN, and Cys-C were determined at baseline by an enzymatic colorimetric method and an automatic analyzer  in our clinical laboratory. We also recorded a range of clinical information, including gender, age, disease duration , history of hypertension, diabetes mellitus, cigarette use, and alcohol abuse. The disease severity of MSA was evaluated by the Unified Multiple System Atrophy Rating Scale (UMSARS) . The dist-test was used to identify differences in continuous variables. The Pearson correlation coefficient was used to determine the associations between serum oxidant factors and disease severity and to identify significant biomarkers in subgroup analysis. The subgroups included MSA with predominant Parkinsonism (MSA-P) and MSA with predominant cerebellar ataxia (MSA-C)  . The Kapn = 28) and MSA-P (n = 24) subgroups; there were no significant differences between these groups with regards to the clinical parameters observed in this study . However, the other three oxidative biomarkers were significantly higher in MSA patients than in healthy controls, as shown in p = 0.023; BUN:5.68 ± 1.67 vs. 4.60 ± 1.24 mmol/L, p < 0.001; Cys-C:0.96 ± 0.15 vs. 0.89 ± 0.14 mg/L, p = 0.024). Next, we compared these significantly elevated serum oxidative biomarkers between the two subgroups. Although we did not identify any significant differences, we found that the MSA-C patients exhibited relatively lower concentrations of these biomarkers than the MSA-P patients, as shown in p = 0.074; BUN:5.44 ± 1.81 vs. 5.95 ± 1.49, p = 0.280; Cys-C:0.92 ± 0.14 vs. 1.00 ± 0.16, p = 0.093).Analysis did not identify a significant difference in the levels of Scr between the MSA patients and healthy subjects, as shown in r = 0.287, p = 0.039), thus suggesting that this factor could act as a potential biomarker for patients with MSA. Next, we investigated the efficacy of serum Cys-C in the MSA-C and MSA-P subgroups. This serum biomarker showed a strong association with the MSA-C subgroup  but not the MSA-P subgroup . In addition, we explored the relationship between serum Cys-C and UMSARS scores in MSA-C patients. This serum biomarker was significantly positive correlated with disease duration  and GDS , but its relation to UMSARS-I , -II , total score  or the disease progression rates  was not statistical significance. These results suggest that serum Cys-C may act as a predictive factor for disease severity in patients with MSA-C.Pearson correlation analyses revealed that there was no significant association between either UA and BUN with the UMSARS score, disease duration, or disease progression rate . The serp = 0.009; 2 = 4.154, p = 0.042). Finally, ROC curve analysis confirmed that this serum biomarker showed good performance as a predictor of disease severity in patients with MSA-C; the area under the curve (AUC) was 0.847  and severe disability (GDS 3–5). We found that MSA-C patients with severe disability had significantly higher concentrations of serum Cys-C than patients with mild disability . Future research should investigate the specific effect of mecobalamin on MSA patients.In conclusion, the serum levels of Cys-C may have a potential to evaluate the disease severity in patients with MSA-C while other variables should be taken into consideration together.Original data for this study are included in the article/The studies involving human participants were reviewed and approved by the independent ethics committees of the First Affiliated Hospital of Chongqing Medical University and the First Affiliated Hospital of Sun Yat-sen University. The patients/participants provided their written informed consent to participate in this study.FY designed the study, draft the manuscript, and contributed to the discussion. TW collected the data, analyzed the data, and contributed to the discussion. HL analyzed the data and draft the manuscript. JL and XW contributed to the discussion. WS designed the study and revised the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.